Identification Of Antigenic Epitopes And Screening Receptor Binding Domain On The Spike Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). During the past 30 years, in spite of preparation of inactivated and live attenuated vaccines, PED has frequently broken out in many swine-raising countries of Asia, resulting in large economic losses. Thus, there is a need to develop novel vaccine of the PEDV, and understand the mechanism of viral entry and immunization. The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a key role in the induction of the neutralizing antibodies and specific receptor binding and cell membrane fusion, because of possessing the neutralizing epitopes and receptor binding domain. Given this, screening and identification of the receptor binding domain and antigenic epitopes were carried out in this study. It is necessary to provide some valuable information for development of diagnosis technology and novel vaccine, and for designing antiviral immunization strategy of PEDV.In order to obtain information about molecular properties of PEDV S gene, the S gene of PEDV strain CV777 adapted to Vero E6 cells was sequenced, using three overlapping cDNA clones which were amplified by RT-PCR based on viral genome RNA. Sequence analysis showed that identity of nucleotides and deduced amino acids was 99.4 % and 99.8 % respectively, comparing with the reference sequence of S gene of PEDV strain CV777. Analysis result of the amino acids hydrophobicity, signal peptide sequence and N-linked glycosylation demonstrated that these molecular properties of the cloned gene S were the same to the parental virus PEDV strain CV777. S protein was divided into the S1 domain (aa 1~789) and S2 domain (aa 790~1383) according to the presence of conserved nonamer and the GxCx motifs at the proteolytic cleavage site of S protein in other members of coronavirus, group II. The S1 domain contains major neutralization epitopes and receptor binding domain of PEDV.To analyze antigenic epitopes of PEDV S protein, the S1 gene targeted libraries containing the major immunodominant region S1 (aa 1~789) of PEDV spike glycoprotein gene were constructed using the fd phage display system which the exogenous polypeptides were expressed in the N-terminal of the fd phage gene III coat protein. The S1 libraries were panned three times with the purified rabbit sera against PEDV. Three short peptides which were displayed by recombinant fd phages showed strong binding affinity with the PEDV antisera, and were designated S1P1 (aa 248~280), S1P2 (aa 442~499) and S1P3 (aa 697~742) respectively. The results of ELISA and Western blot indicated that the GST fusion proteins of the three short peptides were all recognized by the PEDV antisera and S1P3 showed strong binding activity. To further determined antigenicity of the epitopes S1P1, S1P2 and S1P3, the antisera of the GST fusion proteins S1P1, S1P2, S1P3 and S1P123 (fused epitopes of S1P1, S1P2 and S1P3) were prepared. The result of the IFA and ELISA demonstrated that the epitopes S1P2, S1P3 and the fusion epitope S1P123 were able to induce the S1-specific antisera with the binding ability to the native S protein of PEDV cultured in Vero E6 cells. The result showed that the epitopes S1P1, S1P2 and S1P3 were three linear antigenic epitopes on the spike protein of PEDV, and the epitopes S1P2 and S1P3 had good immunogenicity.The S1 domain of PEDV S protein plays a key role in the induction of the neutralizing antibodies. To identify immunodominant region on S protein, the gene encoding its major immunodominant region S1 was amplified by PCR. Four truncated S1 proteins spanning the entire S1 domain fused to GST protein were prepared. These recombinant proteins were designated as S1A-GST, S1B-GST, S1C-GST and S1D-GST. To identify the most important antigenic region of the S1, the truncated S1-GST fusion proteins were examined for their ability to react with immune serum against PEDV and to elicit the formation of neutralization antibodies in immunized animals. It was found that the region S1D (aa 636~789) was able to react with PEDV antiserum and to elicit formation of neutralization antibodies in mice. Moreover, the immune serum against S1D showed the binding ability to the native S protein of PEDV. To accurately identify epitopes on neutralizing epitope region S1D, six specific monoclonal antibodies (McAbs) against the S1D region were prepared. In western blot, these McAbs could react with the native S protein of PEDV. In order to mapping epitopes of the domain S1D, the S1-phage library and Pepscan technology were carried out. The result showed that the McAbs 2C4, 3C3 and 5F8 recognized the epitope S1D5 (aa 744-759), and the McAbs 3G3, 6E6 and 3G5 recognized the epitope S1D6 (aa 756-771). The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 (Y748SNIGVCK755) and SS6 (L764QDGQVKI771) are two core epitopes on the epitopes S1D5 and S1D6, respectively.Information of cellular receptor and its receptor binding region could provide better strategies for developing effective vaccine and medicine. To identify the receptor binding domain of porcine aminopeptidase N (pAPN) receptor of PEDV, the S1 gene targeted libraries of PEDV were panned three times with the soluble pAPN based on standard procedure. Thirty phage clones which selected randomly from the binding-phages of pAPN, were sequenced by the dideoxy method. The result showed that polypeptides displayed by twenty-eight phage clones (excepted two meaningless sequences) were focused on 249~529 amino acids (MRR) on S protein of PEDV. In order to investigate interaction between pAPN and the region MRR of the PEDV S protein, His fusion gene of the region MRR was expressed in insect cells (sf9) using Bac-to-Bac? baculovirus expression system. In Western blot, MRR fusion protein with His tag could react with antiserium of PEDV. However, the further pull-down assay demonstrated that the binding activity of the recombinant protein MRR and pAPN could not be detected in Western blot. These data suggested that the region MRR of the PEDV S protein was a potential receptor binding domain of the porcine aminopeptidase N (pAPN).All together, five linear antigenic epitopes S1P1 (aa 248~280), S1P2 (aa 442~499), S1P3 (aa 697~742), SS5 (aa 748~755) and SS6 (aa 764~771), one neutralizing epitopes (S1D, aa 636~789), and one potential receptor binding domain (MRR, aa 249~529) of the porcine aminopeptidase N (pAPN) were identified on the S protein of PEDV. These data could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of porcine epidemic diarrhea virus and further structure and function analysis of spike protein.